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W M Nauseef

Publications and source records attributed to W M Nauseef.

At least 37 records · Page 2Linked to original sources
PubMed1998

Coordinated participation of calreticulin and calnexin in the biosynthesis of myeloperoxidase.

Myeloperoxidase (MPO) is a neutrophil lysosomal hemeprotein essential for optimal oxygen-dependent microbicidal activity. We have demonstrated previously that calreticulin, a luminal endoplasmic reticulum protein, functions as a molecular chaperone during myeloperoxidase biosynthesis, associating reversibly with the heme-free precursor apopro-MPO. Because the membrane-bound endoplasmic reticulum protein calnexin is structurally and functionally related to calreticulin, we assessed the role of calnexin in myeloperoxidase biosynthesis. Like calreticulin, calnexin coprecipitated exclusively with glycosylated MPO precursors and with apopro-MPO but, in contrast to calreticulin, also with the enzymatically active, heme-containing precursor pro-MPO. To determine if calnexin participated in heme insertion into MPO, we compared the kinetics of chaperone association with MPO precursors using stable transfectants expressing cDNA encoding wild type MPO or mutated forms that do not acquire heme. Transfectants expressing mutant cDNA had prolonged association of MPO-related precursors with calreticulin and especially with calnexin. These studies demonstrate that 1) both calreticulin and calnexin associated with glycosylated apopro-MPO; 2) only calnexin associated selectively with the enzymatically active, heme-containing precursor pro-MPO; and 3) mutants unable to incorporate heme had prolonged association with calnexin. These findings represent the first evidence of a specialized role for calnexin in facilitating protein maturation in the endoplasmic reticulum of myeloid cells.

Calcium-Binding Proteins↗
PubMed1998

Neutrophils exposed to bacterial lipopolysaccharide upregulate NADPH oxidase assembly.

Bacterial LPS is a pluripotent agonist for PMNs. Although it does not activate the NADPH-dependent oxidase directly, LPS renders PMNs more responsive to other stimuli, a phenomenon known as "priming." Since the mechanism of LPS-dependent priming is incompletely understood, we investigated its effects on assembly and activation of the NADPH oxidase. LPS pretreatment increased superoxide (O2-) generation nearly 10-fold in response to N-formyl methionyl leucyl phenylalanine (fMLP). In a broken-cell O2--generating system, activity was increased in plasma membrane-rich fractions and concomitantly decreased in specific granule-rich fractions from LPS-treated cells. Oxidation-reduction spectroscopy and flow cytometry indicated LPS increased plasma membrane association of flavocytochrome b558. Immunoblots of plasma membrane vesicles from LPS-treated PMNs demonstrated translocation of p47-phox but not of p67-phox or Rac2. However, PMNs treated sequentially with LPS and fMLP showed a three- to sixfold increase (compared with either agent alone) in plasma membrane-associated p47-phox, p67-phox, and Rac2, and translocation paralleled augmented O2- generation by intact PMNs. LPS treatment caused limited phosphorylation of p47-phox, and plasma membrane-enriched fractions from LPS- and/or fMLP-treated cells contained fewer acidic species of p47-phox than did those from cells treated with PMA. Taken together, these studies suggest that redistribution of NADPH oxidase components may underlie LPS priming of the respiratory burst.

Cell Membrane↗
PubMed1998

Pattern of inheritance in hereditary myeloperoxidase deficiency associated with the R569W missense mutation.

Myeloperoxidase (MPO) is an essential component of the oxygen-dependent microbicidal system of neutrophils and monocytes. Hereditary deficiency of MPO occurs in 1 in 2,000 to 4,000 individuals in the general population and has been generally considered an autosomal recessive trait. Previous studies have used the peroxidase activity of blood leukocytes to assess the phenotype of affected family members. Eosinophil peroxidase (EPO) also contributes to the peroxidase activity of blood leukocytes. Because EPO expression is normal in MPO-deficient subjects, eosinophil contamination can significantly contribute to peroxidase activity in leukocytes from family members of an MPO-deficient subject and thereby undermine correct interpretation of the inheritance pattern. To avoid this potential problem, we used cytochemical, immunochemical, and genetic techniques to assess the inheritance pattern of MPO deficiency in sixteen individuals from five unrelated kindreds. Each kindred had an index case with MPO deficiency and the R569W missense mutation, a genotype that causes MPO deficiency. Our analysis demonstrated that MPO deficiency was not inherited as a simple autosomal recessive trait. Most subjects were compound heterozygotes with respect to the R569W mutation and demonstrated a spectrum of phenotypes. Our data demonstrate the broad phenotypic impact of compound heterozygosity on the expression and function of a multimeric protein such as MPO.

Female↗
PubMed1998

Insights into myeloperoxidase biosynthesis from its inherited deficiency.

Myeloperoxidase (MPO) is a heme protein present in the granules of neutrophils and monocytes. The activated neutrophil releases MPO into the phagolysosome or into the extracellular space in response to a variety of agonists. During concomitant activation of the NADPH-dependent oxidase, the stimulated neutrophil also generates hydrogen peroxide, and in this way the MPO-hydrogen peroxide-halide system exerts its potent microbicidal activity. Recent interest in MPO has extended well beyond the domain of innate host defense against infection and includes generalized inflammatory diseases, atherosclerosis, and degenerative neurologic diseases. Search of the various data banks using the cDNA sequence for MPO has uncovered previously unsuspected relationships among peroxidatively active proteins in widely different species. In addition, application of the analytical tools of cell and molecular biology has allowed definition of specific genotypes underlying MPO deficiency and the impact of particular mutations on the fate of MPO precursors along the biosynthetic pathway. In parallel, such studies have allowed significant advances in understanding of the normal steps in MPO biosynthesis and intracellular targeting.

Animals↗
PubMed1997

Calreticulin biosynthesis and processing in human myeloid cells: demonstration of signal peptide cleavage and N-glycosylation.

Calreticulin is a soluble endoplasmic reticulum protein comprising the major storage reservoir for inositol trisphosphate-releasable calcium. Although its highly conserved primary structure and a wide range of functions have been well described, less attention has been paid to its biosynthesis, particularly in human tissues. We report analyses of synthesis, proteolytic processing and glycosylation of human calreticulin. In both HL-60 and PLB-985 myeloid cell lines calreticulin was immunoprecipitated as a single 60-kD species without evidence of precursor forms. However, in vitro cell-free synthesis produced a 62-kD primary translation product, which in the presence of microsomal membranes, was processed by cotranslational signal peptide cleavage to a 60-kD species that comigrated with mature calreticulin produced in myeloid cells. Neither tunicamycin treatment of the cells nor endoglycosidase digestion of calreticulin resulted in any forms other than the 60-kD protein on sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis, suggesting that the potential site for N-glycosylation at asparagine-327 was unmodified. However, oxidative derivatization of carbohydrate components with digoxigenin showed that human calreticulin produced in either HL-60 cells or Sf9 insect cells is glycosylated, indicating that glycosylated and nonglycosylated human calreticulin have indistinguishable electrophoretic mobilities. Direct measurement by phenol-H2SO4 confirmed the presence of carbohydrate on recombinant human calreticulin. These data show that human myeloid calreticulin undergoes cotranslational signal peptide cleavage and posttranslational N-linked glycosylation. Although glycosylation of calreticulin has been shown in rat liver and bovine liver and brain, it has been reported to be lacking in other tissues including human lymphocytes.

Animals↗
PubMed1996

Effect of the R569W missense mutation on the biosynthesis of myeloperoxidase.

Human neutrophil microbicidal activity is largely mediated by reactive species generated by the oxygen-dependent myeloperoxidase (MPO) system. Peroxidase-negative neutrophils from many patients with hereditary MPO deficiency possess a 90-kDa MPO-related protein. We recently identified a missense mutation, R569W, in the MPO gene of many subjects with MPO deficiency. In these studies we examined the consequences of R569W on MPO biosynthesis and processing, using stably transfected K562 cells expressing normal MPO or the R569W mutation. K562 cells expressing normal MPO mimicked faithfully many features of MPO biosynthesis in myeloid cells. 1) apopro-MPO was synthesized; 2) a functional heme group was inserted into apopro-MPO, and enzymatically active pro-MPO was thereby generated; 3) pro-MPO underwent proteolytic processing to mature MPO; and 4) hemin augmented the processing of pro-MPO. pREP-R569W cells synthesized apopro-MPO, but heme was not inserted. Neither enzymatically active pro-MPO nor mature MPO was synthesized by transfectants expressing mutated cDNA, confirming our hypothesis that the R569W mutation results in a form of apopro-MPO which does not undergo post-translational processing to enzymatically active MPO species. In addition, these data support previous suggestions that heme insertion into apopro-MPO is necessary for its subsequent proteolytic processing into mature MPO subunits.

Amino Acid Sequence↗
PubMed1995

A domain of p47phox that interacts with human neutrophil flavocytochrome b558.

The NADPH-dependent oxidase of human neutrophils is a multicomponent system including cytosolic and membrane proteins. Activation requires translocation of cytosolic proteins p47phox, p67phox, and Rac2 to the plasma membrane and association with the membrane flavocytochrome b to assemble a functioning oxidase. We report the location of a region in p47phox that mediates its interaction with flavocytochrome b. From a random peptide phage display library, we used biopanning with purified flavocytochrome b to select phage peptides that mimicked potential p47phox binding residues. Using this approach, we identified a region of p47phox from residue 323 to 342 as a site of interaction with flavocytochrome b. Synthetic peptides 315SRKRLSQDAYRRNS328, 323AYRRNSVRFL332, and 334QRRRQARPGPQSPG347 inhibited superoxide (O2-.) production in the broken cell system with IC50 of 18, 57, and 15 microM, respectively. 323AYRRNSVRFL332 and its derivative peptides inhibited phosphorylation of p47phox. However, the functional importance of this peptide was independent of its effects on phosphorylation, since 323AYRRNAVRFL332 inhibited O2-. production, but had no effect on phosphorylation. None of the peptides blocked O2-. production when added after enzyme activation, suggesting that they inhibited the assembly, rather than the activity, of the oxidase. Furthermore these peptides inhibited membrane association of p47phox in the broken cell translocation assay and O2-. production by electropermeabilized neutrophils, thereby supporting the interpretation that this region of p47phox interacts with flavocytochrome b.

Amino Acid Sequence↗
PubMed1995

Calreticulin functions as a molecular chaperone in the biosynthesis of myeloperoxidase.

Myeloperoxidase (MPO), a lysosomal heme protein found exclusively in neutrophils and monocytes, is necessary for efficient oxygen-dependent microbicidal activity. Acquisition of heme by the heme-free MPO precursor apopro-MPO appears to be a prerequisite for its subsequent proteolytic processing and advancement along the biosynthetic pathway to mature MPO. We present data indicating that calreticulin (CRT), a high capacity calcium-binding protein residing in the lumen of the endoplasmic reticulum of a wide variety of cells, interacts specifically with fully glycosylated apopro-MPO. Biosynthetically radiolabeled CRT (60 kDa) and apopro-MPO (90 kDa) were coprecipitated from PLB 985 cells by monospecific antiserum against CRT when the immunoprecipitations were performed either under nondenaturing conditions or following reversible crosslinking. Nonglycosylated MPO precursors synthesized in the presence of tunicamycin did not interact with CRT. The CRT-apopro-MPO interaction was restricted to an early phase of MPO biosynthesis, and CRT did not interact with the later appearing, heme-containing species of MPO, i.e. pro-MPO or the heavy subunit of mature MPO. These data show that CRT participates in the post-translational processing of MPO, perhaps by maintaining apopro-MPO in a conformation competent to accommodate insertion of the heme group. In this general way, CRT shares certain functional properties with the structurally homologous transmembrane calcium-binding endoplasmic reticulum protein calnexin. Both interact with glycosylated biosynthetic precursors of proteins selectively expressed in specialized cells.

Calcium-Binding Proteins↗
PubMed1995

Moesin, ezrin, and p205 are actin-binding proteins associated with neutrophil plasma membranes.

Actin-binding proteins in bovine neutrophil plasma membranes were identified using blot overlays with 125I-labeled F-actin. Along with surface-biotinylated proteins, membranes were enriched in major actin-binding polypeptides of 78, 81, and 205 kDa. Binding was specific for F-actin because G-actin did not bind. Further, unlabeled F-actin blocked the binding of 125I-labeled F-actin whereas other acidic biopolymers were relatively ineffective. Binding also was specifically inhibited by myosin subfragment 1, but not by CapZ or plasma gelsolin, suggesting that the membrane proteins, like myosin, bind along the sides of the actin filaments. The 78- and 81-kDa polypeptides were identified as moesin and ezrin, respectively, by co-migration on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoprecipitation with antibodies specific for moesin and ezrin. Although not present in detectable amounts in bovine neutrophils, radixin (a third and closely related member of this gene family) also bound 125I-labeled F-actin on blot overlays. Experiments with full-length and truncated bacterial fusion proteins localized the actin-binding site in moesin to the extreme carboxy terminus, a highly conserved sequence. Immunofluorescence micrographs of permeabilized cells and cell "footprints" showed moesin co-localization with actin at the cytoplasmic surface of the plasma membrane, consistent with a role as a membrane-actin-linking protein.

Amino Acid Sequence↗
PubMed1995

Clinical evidence of spinal and cerebral histoplasmosis twenty years after renal transplantation.

Disseminated infection with Histoplasma capsulatum frequently involves the nervous system, but the CNS process is generally not clinically apparent. We report an unusual case of a renal transplant recipient with long-standing immunosuppression who presented with clinical evidence of mass lesions in both his cerebral cortex and his spinal cord. Findings of CSF examination were normal, but stereotaxic biopsies of his cortical lesions demonstrated yeast forms and cultures of biopsy specimens yielded H. capsulatum. Clinical defects referable to both the cortical and spinal lesions decreased in severity after the patient received antifungal therapy. Our case illustrates that disseminated histoplasmosis can present in myriad ways and that widespread disease in the CNS can be occult in immunocompromised patients.

Aged↗
PubMed1994

Evidence for a readily dissociable complex of p47phox and p67phox in cytosol of unstimulated human neutrophils.

We explored the association between two cytosolic components of the phagocyte respiratory burst oxidase, p47phox and p67phox. Both of these proteins bound to immobilized GTP or 2',5'-ADP, but not to GDP or ATP. Similarly, triazine dye-ligand chromatography demonstrated coisolation of p47phox and p67phox. Binding of p67phox to GTP was less avid than that of p47phox. Each of the proteins in whole neutrophil cytosol bound separately to GTP in the absence of the other, whereas studies with recombinant proteins showed binding of p47phox but not p67phox. These data suggest that p47phox and p67phox exist as a complex that very likely involves at least one additional cytosolic protein. Sequential precipitation in graded concentrations of ammonium sulfate demonstrated similar profiles for p47phox and p67phox. Charge-based separations using either an anion or cation exchange resin resulted in coelution of the two cytosolic oxidase components, in spite of their widely differing charges as shown by separate analysis of the recombinant proteins. Moreover, preparative isoelectric focusing showed that a portion of the p47phox and p67phox in cytosol coisolated at a pI (7.3-8.3) midway between those of the separate proteins. Despite this, each protein sedimented independently in sucrose density gradients. The data indicate that the complex of p47phox and p67phox was dissociated by increased temperature, high osmolarity, or prolonged incubation. We suggest that under the physiologic conditions in the cytosol of intact phagocytes p47phox, p67phox and probably other proteins exist as a macromolecular complex, the function of which may be to permit en bloc translocation of oxidase components to the plasma membrane.

Ammonium Sulfate↗
PubMed1994

Ceftazidime-related nonconvulsive status epilepticus.

The third-generation cephalosporin, ceftazidime, is widely used for the treatment of serious gram-negative infections. As is true of cephalosporins in general, reported adverse effects have been few. We report a case of ceftazidime-induced status epilepticus in a patient with Pseudomonas aeruginosa meningitis and compare the clinical manifestations of this case with those of two previously described cases of ceftazidime-related encephalopathy. This diagnosis should be entertained and an electroencephalogram should be obtained in all patients with myoclonus and/or altered mental status while they are receiving ceftazidime therapy.

Aged↗
PubMed1994

Hereditary myeloperoxidase deficiency due to a missense mutation of arginine 569 to tryptophan.

Hereditary deficiency of myeloperoxidase (MPO) is a common disorder but its genetic basis is unknown. We have reported that neutrophils from individuals with MPO deficiency lack enzymatic and immunochemical evidence for mature MPO but have a 90-kDa precursor protein. We have thus hypothesized that hereditary MPO deficiency reflects a defect in processing of a mutated primary translation product. Genomic DNA's from normal subjects digested with BglII and probed with radiolabeled cDNA for MPO have a 2.6-kilobase (kb) band. Previously we described the presence of an aberrant 2.1-kb fragment in BglII digests from most individuals with either partial or complete MPO deficiency. We describe here the responsible mutation. The substitution of thymidine for cytosine in exon 10 at nucleotide 8,089 of the genomic sequence results in generation of a recognition site for BglII not present normally and converts the normal 2.6-kb BglII fragment to the 2.1-kb fragment associated with MPO deficiency. At the amino acid level this mutation would replace arginine at codon 569 with tryptophan. Six of seven patients with complete MPO deficiency had this mutation. One subject was homozygous for this mutation whereas five others were heterozygous at this locus. The seventh patient was the only completely deficient subject without this mutation. Thus, at least two mutations and three genotypes can produce the phenotype of MPO deficiency.

Base Sequence↗
PubMed1993

Functional domain in an arginine-rich carboxyl-terminal region of p47phox.

Activation of the neutrophil respiratory burst oxidase involves phosphorylation-dependent translocation of the cytosolic proteins p47phox and p67phox to the plasma membrane, a process in intact cells that is inhibited by staurosporine. We now report that in a cell-free oxidase system, staurosporine and protein kinase C pseudosubstrate PKC(19-36) both inhibited p47phox phosphorylation but had no effect on superoxide generation. In contrast, p47phox phosphorylation, translocation, and superoxide generation were inhibited by a peptide, p47phox(323-332) (AYRRNSVRFL), based on a putative serine phosphorylation domain. This effect was specific for the 323-332 peptide, as it was not observed with two peptides based on other p47phox phosphorylation domains. All three peptides served as substrates for phosphorylation, but the extent of peptide phosphorylation did not correlate with inhibition of oxidase function. p47phox(325-330), which represents the serine phosphorylation motif of p47phox(323-332), did not inhibit translocation or superoxide production despite its ability to block phosphorylation of p47phox. These data indicate the presence of functionally important sites within the p47phox(323-332) peptide. Although serine 328 is in a consensus phosphorylation motif, the lack of correlation in the cell-free system between p47phox phosphorylation and either protein translocation or superoxide formation suggests that a phosphorylation-independent function resides in the 323-332 segment of p47phox.

Amino Acid Sequence↗
PubMed1993

Protein kinase C isotypes and signal-transduction in human neutrophils: selective substrate specificity of calcium-dependent beta-PKC and novel calcium-independent nPKC.

Neutrophils possess at least two phospholipid-dependent forms of protein kinase C, a classical Ca/PS/DG-dependent beta-isotype of protein kinase C and a Ca-independent but PS/DG-dependent novel protein kinase C (nPKC) which we now demonstrate to have different substrate specificities. Activation of human neutrophils triggers assembly of an NADPH oxidase in the membrane and generation of O2-. A role for the major Ca-dependent isotype beta-PKC in neutrophils is proposed in stimulus-induced phosphorylation and association of a cytosolic 47 kDa protein (p47-phox) with the membrane NADPH oxidase. In this study we demonstrate that purified beta-PKC and nPKC have very different substrate specificities; beta-PKC but not nPKC phosphorylated both endogenous and recombinant p47-phox. In addition, beta-PKC but not nPKC phosphorylated [ser25]PKC(19-31), the substrate peptide based on a sequence in the Ca-dependent alpha, beta and gamma-isotypes. Pseudosubstrate(19-36), derived from the C-terminus of Ca-dependent PKC isotypes, inhibited beta-PKC but not nPKC activity using either Histone IIIS or peptide(19-31) as substrate. Pseudosubstrate(19-36) also inhibited beta-PKC catalyzed phosphorylation of endogenous and recombinant p47-phox. Pseudosubstrate(19-36) also inhibited the O2- generation triggered by GTP gamma S in electroporated neutrophils by 50%. 32P-Labelled neutrophils electroporated in the presence of GTP gamma S showed phosphorylation of multiple cytosolic proteins including a 47 kDa band, and phosphorylation of membrane-associated 34 kDa, 47 kDa and 54 kDa proteins. Pseudosubstrate(19-36) inhibited phosphorylation of p47-phox in the membrane but not in the cytosol. These findings suggest translocatable, Ca-dependent isotypes of PKC such as beta-PKC may play a role in the phosphorylation of membrane associated p47-phox and the assembly or maintenance of an active NADPH oxidase.

Calcium↗
PubMed1993

Cytosolic oxidase factors in the NADPH-dependent oxidase of human neutrophils.

The NADPH-dependent superoxide generating system of human PMNs is a complex, multicomponent system. Studies over the past two decades have identified some of the various components both in the membrane and in the cytosol. The cytosolic factors p47 phox and p67 phox are clearly essential components of the oxidase, as evidenced by their absence producing autosomal CGD. Despite this, the specific function of each of these factors in the assembled oxidase remains unknown. In the case of p47 phox, determinants for translocation are multifactorial, depending in part on phosphorylation and in part on the participation of a functional domain at p47 phox(323-332). The importance of SH3 regions and proline-rich domains in intramolecular interactions and associations with the membrane skeleton remain to be defined. In addition, factors which modulate the assembly of this oxidase are largely unknown and their elucidation may provide insights into novel means by which to modify the inflammatory response.

Amino Acid Sequence↗